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102 Chapter 5 5 (25 mg, quant). 1 H NMR (600 MHz, D 2 O) δ 4.86 (1H, s, H-1, Hep-I), 4.47 (1H, d, J = 7.7 Hz, H-1, Gal-II), 4.07 (1H, s, H-2, Hep-I), 4.01 (1H, t, J = 6.5 Hz, H-6, Hep-I), 3.98 – 3.90 (2H, m, H-3, Hep-I; H-4, Hep-I), 3.88 (1H, s, H-4, Gal-II), 3.78 – 3.61 (7H, m, CH 2 , pentyl; H-6, Gal-II; H-5, Gal-II; H-6a, Gal-II; H-3, Gal-II), 3.60 – 3.53 (2H, m, H-5, Hep-I; H-2, Gal- II), 3.52 – 3.46 (1H, m, H-6b, Gal-II), 2.94 (2H, t, J = 7.6 Hz, CH 2 , pentyl), 1.69 – 1.55 (4H, m, 2 x CH 2 , pentyl), 1.46 – 1.33 (2H, m, CH 2 , pentyl). 13 C NMR (151 MHz, D 2 O) δ 100.9 (C-1, Gal-II), 99.3 (C-1, Hep-I), 78.6 (C-3, Hep-I), 75.3 (C-5, Gal-II), 72.6 (C-3, Gal-II), 71.1 (C-5, Hep-I), 70.7 (C-2, Gal-II), 68.7 (C-6, Hep-I), 68.6 (C-4, Gal-II), 67.7 (C-2, Hep-I), 67.3, 64.4 (C-4, Hep-I), 62.6, 61.1, 39.3, 28.0, 26.5, 22.5. ESI HRMS ( m/z ): [M + H] + calcd for C 18 H 35 NO 12 ; 458.2232 found 458.2238 Enzyma�c synthesis Expression of enzymes CgtA and CgtB from Campylobacter jejuni Materials: The full-length codon-op�mized gene encoding for CjCgtB (ENA: CAL35256.1) was synthesized by Genscript and subcloned into pET15b+ (NdeI, BamHI). The same applied to CjCgtA (ENA: AAL05993.1), except the first 30 base pairs (first 10 amino acids) were omi�ed. Bl21(DE3) (C2527H) cells were purchased from New England Biolabs (NEB). Ampicillin (sodium salt) (14417) was ordered through Cayman Chemicals. Imidazole (56750), TRIS-HCl (T5941), NaCl (S9888), lysozyme (62971) and triton X-100 (T8787) came from Sigma-Aldrich. 2xYT medium (BP97432) and 2xYT Agar (BP2466) were ordered from Fisher BioReagents. Isopropyl-beta-D-thiogalactopyranoside (IPTG) (R0393) as well as GelCode Blue Stain Reagent (24592) was ordered from Thermo Scien�fic. The Ni-NTA resin (17-5318-01) and the Superdex 200 Increase 10/300 GL column (28990944) were ordered from GE Healthcare. Sartorius provided Vivaspin 6, 10000 MWCO (VS0602) spinfilters. For SDS-PAGE, SurePAGE, Bis-Tris, 10x8, 4-12%, 12 wells (M00653) from GenScript were used. Laemmli sample buffer 2x (161-0737) and Precision Plus Protein Dual Color Standards (161-0374) were ordered from Bio-Rad Laboratories. A standard buffer consis�ng of 50 mM TRIS-HCl and 250 mM NaCl pH 8 was made. Imidazole was added to the standard buffer at concentra�ons of 20 mM, 50 mM or 250 mM to make wash 1, wash 2 and elu�on buffer (re-adjusted to pH 8 if needed) respec�vely. To wash 1, lysozyme (1mg/mL) and triton X-100 (0.1%) was added to make the lysis buffer. Protocol: BL21(DE3) cells were transformed with each of the individual vectors, a colony was picked from the 2xYT agar plate with ampicillin (100 μg/mL) and expanded to a cell culture volume of 500 mL ampicillin (100 μg/mL) containing 2xYT medium at 37°C. The cells were induced at OD 600 = 0.6 with IPTG (final concentra�on was 1 mM) and cultured overnight at 20°C. Then, cells were pelleted at 3000 xg , resuspended in lysis buffer (5% of culture volume), incubated at 37°C for 1 h and sonicated for 30’ on ice. As His-tag purifica�on of CjCgtA or CjCgtB inherently resulted in loss of ac�vity, clarified lysate was used directly or stored at -20°C for later use. Further characteriza�on of the enzymes was done by SurePage 4-12% gel, using Laemmli sample buffer and the Precision Plus Protein marker as reference.